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mFluor™ Violet 545 SE

<strong>Top)</strong>&nbsp;Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature.&nbsp;<strong>Bottom)</strong>&nbsp;Flow cytometry analysis of whole blood cells stained with CD4-mFluor&trade; Violet 545 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; Violet 545 specific V7-A channel.
<strong>Top)</strong>&nbsp;Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature.&nbsp;<strong>Bottom)</strong>&nbsp;Flow cytometry analysis of whole blood cells stained with CD4-mFluor&trade; Violet 545 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; Violet 545 specific V7-A channel.
<strong>Top)</strong>&nbsp;Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature.&nbsp;<strong>Bottom)</strong>&nbsp;Flow cytometry analysis of whole blood cells stained with CD4-mFluor&trade; Violet 545 conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the mFluor&trade; Violet 545 specific V7-A channel.
Fluorescent dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating dyes to a peptide, protein, antibody, amino-modified oligonucleotide, or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
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Physical properties
Molecular weight983.09
SolventDMSO
Spectral properties
Absorbance (nm)398
Correction Factor (260 nm)1.08
Correction Factor (280 nm)0.496
Extinction coefficient (cm -1 M -1)200001
Excitation (nm)393
Emission (nm)543
Quantum yield0.151
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Molecular weight
983.09
Absorbance (nm)
398
Correction Factor (260 nm)
1.08
Correction Factor (280 nm)
0.496
Extinction coefficient (cm -1 M -1)
200001
Excitation (nm)
393
Emission (nm)
543
Quantum yield
0.151
mFluor™ dyes are developed for multicolor flow cytometry-focused applications. These dyes have large Stokes Shifts, and can be well excited by the laser lines of flow cytometers (e.g., 405 nm, 488 nm and 633 nm). mFluor™ Violet dyes are optimized to be excited with a violet laser at 405 nm. AAT Bioquest offers the largest collection of fluorescent dyes that are excited by violet laser. mFluor™ Violet 545 dyes have fluorescence excitation and emission maxima of ~405 nm and ~530 nm respectively. These spectral characteristics make them a unique color for flow cytometry application. mFluor™ Violet 545 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. mFluor™ Violet 545 SE provides a convenient tool to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) for flow cytometric applications with the violet laser excitation.

Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.
Note     The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M  sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note     The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note     The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.


2. mFluor™ Violet 545 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ Violet 545 SE to make a 10 mM stock solution. Mix well by pipetting or vortex.
Note     Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with mFluor™ Violet 545 SE. You might need further optimization for your particular proteins.
Note     Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.


Run conjugation reaction
  1. Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point:  Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
    Note     We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes. 

Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.
    Note     For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.
    Note     For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of mFluor™ Violet 545 SE to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM101.72 µL508.6 µL1.017 mL5.086 mL10.172 mL
5 mM20.344 µL101.72 µL203.44 µL1.017 mL2.034 mL
10 mM10.172 µL50.86 µL101.72 µL508.6 µL1.017 mL

Molarity calculator

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Spectrum


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Spectral properties

Absorbance (nm)398
Correction Factor (260 nm)1.08
Correction Factor (280 nm)0.496
Extinction coefficient (cm -1 M -1)200001
Excitation (nm)393
Emission (nm)543
Quantum yield0.151

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
mFluor™ Violet 450 SE4064453500010.8110.3380.078
mFluor™ Violet 510 SE4125052500010.8610.4640.366
mFluor™ Violet 540 SE4025351800010.2111.3260.543
mFluor™ Violet 500 SE4105012500010.8110.7690.365
mFluor™ Violet 610 SE4216129000010.310.5320.66
mFluor™ Violet 550 SE5275509000010.3110.4740.306
mFluor™ Violet 505 SE3935044000010.4510.8880.403
mFluor™ Violet 590 SE5645919000010.2210.6320.329
mFluor™ Violet 530 SE393543200001---
mFluor™ Violet 480 SE40447540,000-1.230.6570
mFluor™ Violet 545 Styramide3935432000010.1511.080.496
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References


View all 4 references: Citation Explorer
Methodological considerations for the high sensitivity detection of multiple myeloma measurable residual disease.
Authors: Soh, Kah Teong and Tario, Joseph D and Hahn, Theresa E and Hillengass, Jens and McCarthy, Philip L and Wallace, Paul K
Journal: Cytometry. Part B, Clinical cytometry (2020): 161-173
Lot-to-lot stability of antibody reagents for flow cytometry.
Authors: Böttcher, Sebastian and van der Velden, Vincent H J and Villamor, Neus and Ritgen, Matthias and Flores-Montero, Juan and Escobar, Hugo Murua and Kalina, Tomas and Brüggemann, Monika and Grigore, Georgiana and Martin-Ayuso, Marta and Lecrevisse, Quentin and Pedreira, Carlos E and van Dongen, Jacques J M and Orfao, Alberto
Journal: Journal of immunological methods (2019): 112294
Fluorochrome choices for multi-color flow cytometry.
Authors: Flores-Montero, Juan and Kalina, Tomas and Corral-Mateos, Alba and Sanoja-Flores, Luzalba and Pérez-Andrés, Martin and Martin-Ayuso, Marta and Sedek, Lukasz and Rejlova, Katerina and Mayado, Andrea and Fernández, Paula and van der Velden, Vincent and Bottcher, Sebastian and van Dongen, Jaques J M and Orfao, Alberto
Journal: Journal of immunological methods (2019): 112618
[Diagnostic Value of CD27 Antigen in Patients with Multiple Myeloma].
Authors: Wang, Hui and Liu, Lu and Liu, Fang and Chen, Li-Juan and Qu, Xiao-Yan and Li, Jian-Yong and Wu, Yu-Jie
Journal: Zhongguo shi yan xue ye xue za zhi (2017): 1069-1073